Defective adenoviruses including a therapeutic gene and an immunoprotectove gene

ABSTRACT

Novel adenovirus-derived viral vectors, the preparation thereof, and the use of such vectors in gene therapy, are disclosed. In particular, defective adenoviruses having a genome that includes a first recombinant DNA containing a therapeutic gene and a second recombinant DNA containing an immunoprotective gene, are disclosed.

This application is a 371 of International Application PCT/FR95/01326,filed Oct. 11, 1995, and which designated the United States.

The present invention relates to new viral vectors, to their preparationand to their use in gene therapy. It also relates to pharmaceuticalcompositions containing the said viral vectors. More especially, thepresent invention relates to recombinant adenoviruses as vectors forgene therapy.

Gene therapy consists in correcting a deficiency or an abnormality(mutation, aberrant expression, and the like) by introducing geneticinformation into the cell or organ affected. This genetic informationmay be introduced either in vitro into a cell extracted from the organ,the modified cell then being reintroduced into the body, or directly invivo into the appropriate tissue. In this second case, differenttechniques exist, including various techniques of transfection involvingcomplexes of DNA and DEAE-dextran (Pagano et al., J.Virol. 1 (1967)891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989)375) and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), theuse of liposomes (Fraley et al., J.Biol.Chem. 255 (1980) 10431), and thelike. More recently, the use of viruses as vectors for gene transfer hasbeen seen to be a promising alternative to these physical transfectiontechniques. In this connection, different viruses have been tested fortheir capacity to infect certain cell populations. This appliesespecially to retroviruses (RSV, HMS, MMS, and the like), the HSV virus,adeno-associated viruses and adenoviruses.

Among these viruses, the adenoviruses display certain properties whichare advantageous for use in gene therapy. In particular, they have afairly broad host range, are capable of infecting resting cells and donot integrate in the genome of the infected cell. Adenoviruses arelinear, double-stranded DNA viruses approximately 36 kb in size. Theirgenome comprises, in particular, an inverted repeat sequence (ITR) attheir end, an encapsidation sequence, early genes and late genes (seeFIG. 1). The main early genes are the E1 (E1a and E1b), E2, E3 and E4genes. The main late genes are the L1 to L5 genes.

In view of the properties of adenoviruses, mentioned above, the latterhave already been used for in vivo gene transfer. To this end, differentvectors derived from adenoviruses have been prepared, incorporatingdifferent genes (β-gal, OTC, α-1AT, cytokines, and the like). In each ofthese constructions the adenovirus has been modified so as to render itincapable of replication in the infected cell. Thus, the constructionsdescribed in the prior art are adenoviruses from which the E1 (E1aand/or E1b) and, where appropriate, E3 regions have been deleted, inwhich regions a heterologous DNA sequence is inserted(Levrero et al.,Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161).

However, as in the case of all known viruses, the administration of awild-type adenovirus induces a substantial immune response (Routes etal., J. Virol 65 (1991) 1450). This immunogenicity has also beenobserved following the administration of recombinant adenoviruses whichare defective for replication (Yang et al., PNAS (1994) 4407). One ofthe major roles of the immune system consists, in effect, in destroyingnon-self or altered-self elements. The administration of a gene therapyfactor of adenoviral origin introduces non-self units into the body.Similarly, cells infected with such a vector and expressing, as aresult, an exogenous therapeutic gene become altered-self elements.Hence it is normal for the immune system to react against these vectorsand infected cells as if they were foreign bodies. This immune responseto infected cells constitutes a major obstacle to the development ofthese viral vectors, since (i) by inducing a destruction of the infectedcells, it limits the period of expression of the therapeutic gene andhence the therapeutic effect, (ii) it induces a substantial concomitantinflammatory response, and (iii) it brings about rapid elimination ofthe infected cells after repeated injections. Thus, the expression ofβ-galactosidase encoded by a recombinant adenovirus administered in themuscle of immunocompetent mice is reduced to minimum levels 40 daysafter injection (Kass-Eisler et al., PNAS 90 (1993) 11498). Similarly,the expression of gene transferred by adenoviruses into the liver issignificantly reduced after 4 months (Li et al., Hum. Gene Ther. 4(1993) 403), and the expression of factor IX transferred by adenovirusesinto hepatocytes of haemophilic dogs disappears 100 days after injection(Kay et al., PNAS 91 (1994) 2353).

Hence it would appear that the exploitation of vectors derived fromadenoviruses in gene therapy entails the possibility of reducing theimmune response to these vectors or the infected cells. This constitutesspecifically the subject of the present invention. The present inventionrelates, in effect, to new vectors derived from adenoviruses displayingan immunogenicity which is greatly reduced or even eliminated. Thevectors of the invention are hence especially suitable for gene therapyapplications, in particular in man.

A first subject of the present invention relates to a defectiveadenovirus whose genome comprises a first recombinant DNA containing atherapeutic gene and a second recombinant DNA containing animmunoprotective gene.

The present invention is partly the outcome of the demonstration that itis possible to incorporate several genes of interest in adenoviruses,and to obtain a substantial expression of these different genes in theinfected cells. The present invention is also the outcome of theconstruction of adenoviral vectors capable of incorporating severaltherapeutic genes under conditions permitting their optimal expression.It is also the outcome of the demonstration that coexpression in theinfected cell of certain genes is capable of inducing animmunoprotective effect, and thus of leading the vectors of theinvention and/or the infected cells to evade the immune system. Thepresent invention thus provides viral vectors displaying immunologicaland therapeutic properties which are thoroughly advantageous for thepurpose of their use in gene or cell therapy.

The recombinant DNAs according to the present invention are DNAfragments containing the gene in question (therapeutic orimmunoprotective) and optionally signals permitting its expression,constructed in vitro and then inserted into the adenovirus genome. Therecombinant DNAs used in the context of the present invention can becomplimentary DNAs (cDNA), genomic DNAs (gDNA) or hybrid constructionsconsisting, for example, of a cDNA into which one or more introns mightbe inserted. They can also be synthetic or semi-synthetic sequences.These DNAs may be of human, animal, vegetable, bacterial, viral, and thelike, origin. It is especially advantageous for cDNAs or gDNAs to beused.

The insertion of the genes in question in the form of recombinant DNAsaccording to the invention affords greater flexibility in theconstruction of the adenoviruses, and permits better control of theexpression of the said genes.

Thus, the recombinant DNAs, (and hence the two genes of interest)incorporated in the adenoviral vectors according to the invention may beorganized in different ways.

In the first place, they may be inserted at the same site of theadenovirus genome or at selected, different sites. In particular, therecombinant DNAs may be inserted at least partially in the E1, E3 and/orE4 regions of the adenovirus genome, replacing or in addition to viralsequences.

Next, they may each contain a transcription promoter, which may beidentical or different. This configuration enables higher levels ofexpression to be obtained, and affords better control of the expressionof the genes. In this case, the two genes may be inserted in the sameorientation or in opposite orientations.

They may also constitute a single transcriptional entity. In thisconfiguration the two recombinant DNAs are adjacent and positioned insuch a way that both genes are under the control of a single promoterand give rise to a single premessenger RNA. This arrangement isadvantageous since it enables a single transcription promoter to beused.

Lastly, the use of recombinant DNAs according to the invention enablestranscription promoters of different natures to be used, and inparticular strong or weak, regulated or constituted, tissue-specific orubiquitous, and the like, promoters.

The choice of expression signals and of the respective position of therecombinant DNAs is especially important for obtaining a high expressionof the therapeutic gene and a substantial immunoprotective effect.

As a therapeutic gene which may be used for the construction of thevectors of the present invention, any gene coding for a product having atherapeutic effect may be mentioned. The product thus encoded can be aprotein, a peptide, an RNA, and the like.

In the case of a proteinaceous product, this can be homologous withrespect to the target cell (that is to say a product which is normallyexpressed in the target cell when the latter does not display anypathology). In this case, the expression of a protein makes it possible,for example, to compensate for an insufficient expression in the cell orfor the expression of a protein that is inactive or poorly active as aresult of a modification, or alternatively to overexpress the saidprotein. The therapeutic gene can also code for a mutant of a cellularprotein, having enhanced stability, modified activity, and the like. Theproteinaceous product can also be heterologous with respect to thetarget cell. In this case, an expressed protein can, for example,supplement or supply an activity which is deficient in the cell,enabling it to combat a pathology, or stimulate an immune response.

Among proteinaceous products which are therapeutic for the purposes ofthe present invention, there may be mentioned, more especially, enzymes,blood derivatives, hormones, lymphokines, namely interleukins,interferons, TNF, and the like (FR 92/03120), growth factors,neurotransmitters or their precursors or synthetic enzymes, trophicfactors, namely BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NTS,HARP/pleiotrophin, and the like; apolipoproteins, namely ApoAI, ApoAIV,ApoE, and the like (FR 93/05125), dystrophin or a minidystrophin (FR91/11947), the CFTR protein associated with cystic fibrosis,tumour-suppressing genes, namely p53, Rb, RaplA, DCC, k-rev, and thelike (FR 93/04745), genes coding for factors involved in coagulation,namely factors VII, VIII, IX, genes participating in DNA repair, and thelike.

As mentioned above, the therapeutic gene can also be an antisense geneor sequence, the expression of which in the target cell enables theexpression of cellular genes or the transcription of cellular mRNA to becontrolled. Such sequences can, for example, be transcribed in thetarget cell into RNAs complementary to cellular mRNAs, and can thusblock their translation into protein, according to the techniquedescribed in Patent EP 140,308. Antisense sequences also includesequences coding for ribozymes, which are capable of selectivelydestroying target RNAs EP 321,201).

As mentioned above, the therapeutic gene can also contain one or moregenes coding for an antigenic peptide capable of generating an immuneresponse in humans or animals. In this particular embodiment, theinvention hence makes it possible to produce either vaccines orimmunotherapeutic treatments applied to humans or to animals, inparticular against microorganisms, viruses or cancers. Such antigenicpeptides can be, in particular, specific to the Epstein-Barr virus, theHIV virus, the hepatitis B virus (EP 185,573) or the pseudorabies virus,or alternatively tumour-specific (EP 259,212). In this embodiment, noimmune response will be generated against the vector virus or theinfected cell, but the selected antigen will be produced and will alonebe capable of being immunogenic.

The therapeutic genes may be of human, animal, vegetable, bacterial,viral, and the like, origin. They may be obtained by any technique knownto a person skilled in the art, and in particular by the screening oflibraries, by chemical synthesis or alternatively by mixed methodsincluding chemical or enzymatic modification of sequences obtained bythe screening of libraries.

The immunoprotective gene used in the context of the present inventioncan be of different types. More preferably, the Applicant has now shownthat the use of a gene whose product affects the activity of the majorhistocompatibility complex (MHC) or affects cytokine activity makes itpossible to reduce considerably, or even to eliminate, any immunereaction against the vector or the infected cells. The vectors therebyobtained are especially advantageous, since they possess muchlonger-lasting action in vivo and hence a greater therapeutic effect,lack an inflammatory and immunogenic effect and may be used with areduced number of injections.

Antigen presenting cells present antigenic peptides at their surface, incombination with molecules of the major histocompatibility complex classI (MHC-I). The receptors of cytotoxic T cells (CTL) recognize thecomplexes formed between the said MHC class I molecules and the saidantigenic peptides. This recognition induces cell death by CTL. TheApplicant has now shown that it is possible to coexpress in anadenoviral vector a therapeutic gene and a gene capable of impairing theexpression of MHC-I molecules, and that this coexpression produces alasting therapeutic effect without immune or inflammatory reactions.Among genes whose product affects the activity of the majorhistocompatibility complex, it is preferable to use in the context ofthe invention genes whose product at least partially inhibits theexpression of the MHC proteins or antigen presentation. As preferredexamples, certain genes contained in the adenovirus E3 region, theherpes virus ICP47 gene or the cytomegalovirus UL18 gene may bementioned.

The E3 region of the adenovirus genome contains different reading frameswhich, by alternative splicing, give rise to different proteins. Amongthe latter, the Gp19k (or E3−19k) protein is a glycosylatedtransmembrane protein localized in the membrane of the endoplasmicreticulum (ER). This protein comprises a luminal domain binding MHC-Imolecules and a C-terminal cytoplasmic end capable of bindingmicrotubules (or tubulin), which acts to anchor the gp19k protein in themembrane of the ER. Gp19k is thus capable of preventing the expressionof the MHC-I molecules at the cell surface by interaction andsequestration in the ER. However, in the absence of viral replication,the gp19k protein is weakly expressed by adenoviruses. The nativepromoter contains, in effect, some regulatory elements, such as bindingelements of the NF-kB type, which limit the conditions for expression ofthis protein. Moreover, the expression of gp19k is also dependent on theproduction of an alternative splicing. Introduction into the vectors ofthe invention of a recombinant DNA containing a sequence (preferablycDNA) coding for gp19k enables the expression of the said protein to becontrolled and optimized. In particular, the use of constitutedpromoters and the elimination of the other reading frames makes itpossible to increase greatly the expression of this protein and toescape from the dependence on viral replication and the presence ofinducing elements. This makes it possible in an especially advantageousway to decrease considerably the lysis of the infected cells by CTL andthus to increase and prolong the in vivo production of the therapeuticgene. The examples in the present application describe, in particular,the construction of a defective adenovirus carrying a recombinant DNAcomprising a marker gene under the control of the RSV promoter, and asecond recombinant DNA carrying a sequence coding for the gp19k proteinunder the control of the RSV constitutive promoter (Ad-βgal-gp19k). Theresults presented demonstrate that cells infected with this vectorexpress β-galactosidase at levels which are as high as cells infectedwith an adenovirus containing only the β-gal gene. This shows that thepresence of the second recombinant DNA does not affect the levels ofexpression of the first one. Next, the results presented show that cellsinfected with the Ad-βgal-gp19k adenovirus are protected against lysisby CTL, which is not the case with cells infected with an Ad-βgaladenovirus. Furthermore, the presence of the second recombinant DNA inthe vectors of the invention inhibits the clonal expansion oflymphocytes directed against the infected cells. The vectors of theinvention hence induce a significant reduction in the immune response bythe CTL to the infected cells.

Other proteins encoded by the E3 region of the adenovirus genome, suchas the 10.4k and 14.5k proteins, display some properties which areadvantageous for the purpose of their incorporation in the vectors ofthe invention.

The herpes simplex virus ICP47 gene constitutes another immunoprotectivegene which is especially advantageous for the purposes of the presentinvention. Cells infected with the herpes simplex virus display aresistance to CTL-induced lysis. It has been shown that this resistancecould be conferred by the ICP47 gene, which is capable of reducing theexpression of the MHC-I molecules at the cell surface. Incorporation ofthe ICP47 gene in a recombinant DNA according to the invention alsoenables the recombinant viruses of the invention to evade the immunesystem.

The cytomegalovirus UL18 gene constitutes another preferred example ofan immunoprotective gene according to the invention. The UL18 geneproduct is capable of binding β₂-microglobulin (Browne et al. Nature 347(1990) 770). β₂-Microglobulin is one of the chains of MHC-I molecules.Incorporation of the UL18 gene in a recombinant DNA according to theinvention thus makes it possible to decrease the number of functionalβ₂-microglobulin molecules in the cells infected with the viruses of theinvention, and hence to decrease the capacities of these cells toproduce complete and functional MHC-I molecules. This type ofconstruction hence enables the infected cells to be protected from lysisby CTL.

As mentioned above, the immunoprotective gene used in the context of thepresent invention is, in another preferred embodiment, a gene whoseproduct affects the activity or the pathways of signalling of cytokines.The cytokines constitute a family of secreted proteins which act assignalling molecules for the immune system. They can attract the cellsinvolved in immunity, activate them, induce their proliferation and evenact directly on the infected cells to kill them.

Among the genes whose product affects the activity or the pathways ofsignalling of cytokines, there may be mentioned the genes participatingin the synthesis of cytokines, or whose product is capable ofsequestering cytokines, of antagonizing their activity or of interferingwith the intercellular signalling pathways. As preferred examples,special mention may be made of the Epstein-Barr virus BCRF1 gene, thecowpox virus crmA and crmB genes, the vaccinia virus B15R and B18Rgenes, the cytomegalovirus US28 gene and the adenovirus E3-14.7, E3-10.4and E3-14.5 genes.

The vaccinia virus B15R gene codes for a soluble protein capable ofbinding interleukin-1β (the secreted form of interleukin-1), and thus ofpreventing this cytokine from binding to its cell receptors.Interleukin-1 is, in effect, one of the first cytokines produced inresponse to an antigen attack, and it plays a very important part in thesignalling of the immune system at the beginning of infection. Thepossibility of incorporating the B15R gene in a vector according to theinvention advantageously enables IL-1β activity to be reduced, inparticular as regards the activation of immune cells, and as a resultenables the cells infected with the viruses of the invention to beprotected locally against a substantial immune response. Geneshomologous with the B15R gene may also be used, such as the cowpoxvirus—gene.

In the same way, the vaccinia virus B18R gene codes for a proteinhomologous with the interleukin-6 receptor. This gene, or any functionalhomologue, may also be used in the vectors of the invention to inhibitthe binding of interleukin-6 to its cell receptor and thus to reduce theimmune response locally.

Still in the same way, the cowpox virus crmB gene may be advantageouslyused. This gene codes, in effect, for a secreted protein capable ofbinding TNF and of competing with the TNF receptors at the cell surface.Hence this gene makes it possible, in the viruses of the invention, todecrease locally the concentration of active TNF capable of destroyingthe infected cells. Other genes coding for proteins capable of bindingTNF and of at least partially inhibiting its binding to its receptorsmay also be used.

The cowpox virus crmA gene, for its part, codes for a protein having aprotease inhibitor activity of the spermine type, which is capable ofinhibiting the synthesis of interleukin-1. This gene may hence be usedto decrease the concentration of interleukin-1 locally and thus toreduce the development of the immune and inflammatory response.

The Epstein-Barr virus BCRF1 gene codes for an analogue ofinterleukin-10. The product of this gene is a cytokine capable ofdecreasing the immune response and of changing its specificity whileinducing the proliferation of B lymphocytes.

The cytomegalovirus US28 gene codes for a protein homologous with thereceptor for macrophage inflammatory protein 1α (MIP-1α). This proteinis hence capable of acting as a competitor for the MIP receptors, andhence of inhibiting its activity locally.

The product of the adenovirus E3-14.7, E3-10.4 and E3-14.5 genes iscapable of blocking the transmission of the intercellular signalmediated by certain cytokines. When the cytokines bind to their receptorat the surface of an infected cell, a signal is transmitted to thenucleus to induce cell death or stop protein synthesis. This isespecially the case with tumour necrosis factor (TNF). Incorporation ofthe E3-14.7, E3-10.4 and/or E3-14.5 genes in a recombinant DNA accordingto the invention for the purpose of their constitutive or regulatedexpression enables TNF-induced intercellular signalling to be blocked,and thus enables the cells infected with recombinant viruses of theinvention to be protected from the toxic effects of this cytokine.

A local and transient inhibition may be especially advantageous. It maybe obtained, in particular, by the choice of the particular expressionsignals (cytokine-dependent promoters for example), as described below.

It should be understood that other homologous genes or genes havingsimilar functional properties may be used for the construction of thevectors of the invention. These different genes may be obtained by anytechnique known to a person skilled in the art, and in particular by thescreening of libraries, by chemical synthesis or alternatively by mixedmethods including the chemical or enzymatic modification of sequencesobtained by the screening of libraries. In addition, these differentgenes may be used alone or in combination(s).

One of the other important aspects of the present invention relates tothe choice of transcription promoters used for directing the expressionof the genes. As mentioned above, it may be especially important to usea promoter capable of constitutively expressing the gene placed underits control. This is the case, for example, with the gp19k gene or ahomologue, if it is desired to obtain substantial immunoprotection. Incontrast, to control the expression of an immunoprotective geneaffecting cytokine activity, a regulated expression may be desirable. Asregards the expression of the therapeutic gene, the choice of expressionsignals depends on the nature of the therapeutic product, the pathologyin question and the tissue targeted.

The promoters which may be used for the construction of the recombinantDNAs of the invention can be the promoters which are naturallyresponsible for the expression of the therapeutic or immunoprotectivegene in question when they are capable of functioning in the infectedcell. However, they are preferably sequences of different origin(responsible for the expression of other proteins, or even synthetic),especially for controlling the expression of the immunoprotective gene.In particular, they can be promoter sequences of eukaryotic or viralgenes. For example, they can be promoter sequences originating from thegenome of the cell which it is desired to infect. Similarly, they can bepromoter sequences originating from the genome of a virus, including theadenovirus used. In this connection, the promoters of the E1A, MLP, CMV,RSV, and the like, genes may be mentioned for example. In addition,these expression sequences may be modified by the addition of activatoror regulatory sequences or sequences permitting a tissue-specificexpression. Moreover, when the recombinant DNA does not containexpression sequences, it may be inserted into the genome of thedefective virus downstream of such a sequence. A preferred promoter forthe production of the vectors of the invention consists of the Roussarcoma virus LTR (RSVLTR). Since this promoter is constitutive andstrong, it enables substantial immunoprotection to be induced by gp19k.Mammalian promoters may also be of great interest, such as the promoterof the PGK, albumin, and the like, genes. It can be especiallyadvantageous to use regulated or tissue-specific promoters so as to beable to target the synthesis of the therapeutic and/or immunoprotectiveproducts. In particular, for the expression of an immunoprotective geneinhibiting cytokine activity, it can be especially advantageous to usean inducible promoter in order to obtain a localized effect. Induciblepromoters are, for example, cytokine-induced promoters, so that theimmunoprotective effect takes place only in response to an immunereaction.

Moreover, the recombinant DNA can also contain a signal sequencedirecting the synthesized product into the pathways of secretion of thetarget cell. This signal sequence can be the natural signal sequence ofthe gene in question (therapeutic or immunoprotective) whereappropriate, but it can also be any other functional signal sequence oran artificial signal sequence.

As mentioned above, different configurations may be envisaged for theproduction of the vectors of the invention. The vectors of the inventioncan, in the first place, contain the two genes in the form of a singletranscriptional entity. In this configuration, the two recombinant DNAsare adjacent, arranged in such a way that both genes are under thecontrol of a single promoter and give rise to a single premessenger RNA.This configuration is advantageous since it enables a singletranscription promoter to be used to regulate the expression of bothgenes. Moreover, this single transcriptional entity may be incorporatedin the adenoviral vector in both possible orientations.

Advantageously, both recombinant DNAs contain their own transcriptionpromoter. This configuration enables higher levels of expression to beobtained, and affords better control of the expression of the genes. Inthis case, the two recombinant DNAs may be inserted in the sameorientation or in opposite orientations, in the same site of theadenovirus genome or at different sites.

Preferably, the recombinant DNAs are inserted at least partially in theE1, E3 or E4 regions of the adenovirus genome. When they are inserted attwo different sites, it is preferable in the context of the invention touse the E1 and E3 or E1 and E4 regions. The examples show, in effect,that this organization permits a high expression of both genes withoutinterference between the two. Advantageously, the recombinant DNAs areinserted as a replacement for viral sequences.

An especially preferred embodiment of the present invention consists ofa defective adenovirus containing a first recombinant DNA containing atherapeutic gene and a second recombinant DNA containing animmunoprotective gene, in which the two recombinant DNAs are inserted inthe E1 region.

An especially preferred embodiment of the present invention consists ofa defective adenovirus containing a first recombinant DNA containing atherapeutic gene inserted in the E1 region, and a second recombinant DNAcontaining an immunoprotective gene inserted in the E3 region.

As mentioned above, the adenoviruses of the present invention aredefective, that is to say they are incapable of replicating autonomouslyin the target cell. Generally, the genome of the defective adenovirusesaccording to the present invention hence lacks at least the sequencesneeded for replication of the said virus in the infected cell. Theseregions may be either removed (wholly or partially), or renderednon-functional, or replaced by other sequences, and in particular by thetherapeutic genes. The defective character of the adenoviruses of theinvention is an important feature, since it ensures thenon-dissemination of the vectors of the invention after administration.

In a preferred embodiment, the adenoviruses of the invention comprisethe ITR sequences and a sequence permitting encapsidation, and possess adeletion of all or part of the E1 gene.

The inverted repeat sequences (ITR) constitute the origin of replicationof the adenoviruses. They are localized at the 3′ and 5′ ends of theviral genome (see FIG. 1), from where they may be isolated readilyaccording to the traditional techniques of molecular biology known to aperson skilled in the art. The nucleotide sequence of the ITR sequencesof human adenoviruses (especially of the serotypes Ad2 and Ad5) isdescribed in the literature, as well as those of canine adenoviruses (inparticular CAV1 and CAV2). As regards the Ad5 adenovirus for example,the left-hand ITR sequence corresponds to the region comprisingnucleotides 1 to 103 of the genome.

The encapsidation sequence (also designated Psi sequence) is needed forencapsidation of the viral DNA. This region must hence be present inorder to permit the preparation of defective recombinant adenovirusesaccording to the invention. The encapsidation sequence is localized inthe genome of the adenoviruses, between the left-hand (5′) ITR and theE1 gene (see FIG. 1). It may be isolated or synthesized artificially bytraditional techniques of molecular biology. The nucleotide sequence ofthe encapsidation sequence of human adenoviruses (especially of theserotypes Ad2 and Ad5) is described in the literature, as well as thoseof canine adenoviruses (in particular CAV1 and CAV2). As regards the Ad5adenovirus for example, the encapsidation sequence corresponds to theregion comprising nucleotides 194 to 358 of the genome.

More preferably, the adenoviruses of the invention comprise the ITRsequences and a sequence permitting encapsidation, and possess adeletion of all or part of the E1 and E4 genes.

In an especially preferred embodiment, the genome of the adenovirusesaccording to the invention carries a deletion of all or part of the E1,E3 and E4 genes, and still more preferably of all or part of the E1, E3,L5 and E4 genes.

The adenoviruses of the invention may be prepared from adenoviruses ofdiverse origins. There are, in effect, different serotypes ofadenovirus, the structure and properties of which vary somewhat butwhich display a comparable genetic organization. Thus, the teachingsdescribed in the present application may be readily reproduced by aperson skilled in the art for any type of adenovirus.

More especially, the adenoviruses of the invention may be of human,animal or mixed (human and animal) origin.

As regards adenoviruses of human origin, it is preferable to use thoseclassified in group C. More preferably, among the different serotypes ofhuman adenovirus, it is preferable to use adenoviruses type 2 or 5 (Ad2or Ad5) in the context of the present invention.

As mentioned above, the adenoviruses of the invention may also be ofanimal origin, or may contain sequences originating from adenoviruses ofanimal origin. The Applicant has, in effect, shown that adenoviruses ofanimal origin are capable of infecting human cells with great efficacy,and that they are incapable of propagating in the human cells in whichthey have been tested (see Application FR 93/05954). The Applicant hasalso shown that adenoviruses of animal origin are in no waytrans-complemented by adenoviruses of human origin, thereby eliminatingany risk of recombination and propagation in vivo in the presence of ahuman adenovirus, which can lead to the formation of an infectiousparticle. The use of adenoviruses or of regions of adenoviruses ofanimal origin is hence especially advantageous, since the risks inherentin the use of viruses as vectors in gene therapy are even lower.

The adenoviruses of animal origin which may be used in the context ofthe present invention can be of canine, bovine, murine (for example:Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian oralternatively simian (for example: SAV) origin. More especially, amongavian adenoviruses, there may be mentioned the serotypes 1 to 10 whichare available in the ATCC, such as, for example, the strains Phelps(ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR-827), IBH-2A (ATCCVR-828), J2-A (ATCC VR-829), T8-A (ATCC VR-830), K-11 (ATCC VR-921) oralternatively the strains referenced ATCC VR-831 to 835. Among bovineadenoviruses, the different known serotypes may be used, and inparticular those available in the ATCC (types 1 to 8) under thereferences ATCC VR-313, 314, 639-642, 768 and 769. There may also bementioned murine adenoviruses FL (ATCC VR-550) and E20308 (ATCC VR-528),ovine adenovirus type 5 (ATCC VR-1343) or type 6 (ATCC VR-1340), porcineadenovirus 5359), or simian adenoviruses such as, in particular, theadenoviruses referenced in the ATCC under the numbers VR-591-594,941-943, 195-203, and the like.

Among the different adenoviruses of animal origin, it is preferable inthe context of the present invention to use adenoviruses or regions ofadenoviruses of canine origin, and in particular all strains of CAV2adenoviruses [strain Manhattan or A26/62 (ATCC VR-800) for example].Canine adenoviruses have been subjected to many structural studies.Thus, complete restriction maps of CAV1 and CAV2 adenoviruses have beendescribed in the prior art (Spibey et al., J. Gen. Virol. 70 (1989)165), and the E1a and E3 genes as well as the ITR sequences have beencloned and sequenced (see, in particular, Spibey et al., Virus Res. 14(1989) 241; Linne, Virus Res. 23 (1992) 119, WO 91/11525).

The defective recombinant adenoviruses according to the invention may beprepared in different ways.

A first method consists in transfecting the DNA of the defectiverecombinant virus prepared in vitro (either by ligation or in plasmidform) into a competent cell line, that is to say one carrying in transall the functions needed for complementation of the defective virus.These functions are preferably integrated in the genome of the cell,thereby enabling risks of recombination to be avoided and endowing thecell line with enhanced stability.

A second approach consists in cotransfecting the DNA of the defectiverecombinant virus prepared in vitro (either by ligation or in plasmidform) and the DNA of a helper virus into a suitable cell line. Accordingto this method, it is not necessary to have at one's disposal acompetent cell line capable of complementing all the defective functionsof the recombinant adenovirus. A part of these functions is, in effect,complemented by the helper virus. This helper virus must itself bedefective, and the cell line carries in trans the functions needed forits complementation. Among cell lines which may be used, in particular,in the context of this second approach, the human embryonic kidney line293, KB cells, Hela, MDCK, GHK cells, and the like, may be mentioned inparticular (see examples).

Thereafter, the vectors which have multiplied are recovered, purifiedand amplified according to traditional techniques of molecular biology.

According to a variant of embodiment, it is possible to prepare invitro, either by ligation or in plasmid form, the DNA of the defectiverecombinant virus carrying the appropriate deletions and the tworecombinant DNAs. As mentioned above, the vectors of the inventionadvantageously possess a deletion of all or part of certain viral genes,in particular the E1, E3, E4 and/or L5 genes. This deletion maycorrespond to any type of elimination affecting the gene in question. Itmay correspond, in particular, to the elimination of all or part of thecoding region of the said gene, and/or of all or part of the regionpromoting transcription of the said gene. The elimination is generallycarried out on DNA of the defective recombinant virus, for example bydigestion by means of suitable restriction enzymes, followed byligation, according to the techniques of molecular biology, asillustrated in the examples. The recombinant DNAs may then be insertedinto this DNA by enzymatic cleavage, followed by ligation in selectedregions and in the chosen orientation.

The DNA thereby obtained, which hence carries the appropriate deletionsand both recombinant DNAs, enables the defective recombinant adenoviruscarrying the said deletions and recombinant DNAs to be generateddirectly. This first variant is particularly suited to the production ofrecombinant adenoviruses in which the genes are arranged in the form ofa single transcriptional unit, or under the control of separatepromoters but inserted at the same site of the genome.

It is also possible to prepare the recombinant virus in two steps,permitting the successive introduction of the two recombinant DNAs.Thus, the DNA of a first recombinant virus carrying the appropriatedeletions (or a part of the said deletions) and one of the recombinantDNAs is constructed by ligation or in plasmid form. This DNA is thenused to generate a first recombinant virus carrying the said deletionsand a recombinant DNA. The DNA of this first virus is then isolated andcotransfected with a second plasmid or the DNA of a second defectiverecombinant virus carrying the second recombinant DNA, the appropriatedeletions (portion not present on the first virus) and a regionpermitting homologous recombination. This second step thus generates thedefective recombinant virus carrying the two recombinant DNAs. Thisvariant of preparation is especially suitable for the preparation ofrecombinant viruses carrying two recombinant DNAs inserted at twodifferent regions of the adenovirus genome.

The present invention also relates to any pharmaceutical compositioncomprising one or more defective adenoviruses as described above. Thepharmaceutical compositions of the invention may be formulated with aview to topical, oral, parenteral, intranasal, intravenous,intramuscular, subcutaneous, intraocular, transdermal, and the like,administration.

Preferably, the pharmaceutical composition contains vehicles which arepharmaceutically acceptable for an injectable formulation. These can be,in particular, sterile, isotonic saline solutions (monosodium ordisodium phosphate, sodium, potassium, calcium or magnesium chloride,and the like, or mixtures of such salts), or dry, in particularlyophilized, compositions which, on adding sterilized water orphysiological saline, as the case may be, enable injectable solutions tobe formed.

The doses of virus used for the injection may be adapted in accordancewith different parameters, and in particular in accordance with the modeof administration used, the pathology in question, the gene to beexpressed or the desired period of treatment. Generally speaking, therecombinant adenoviruses according to the invention are formulated andadministered in the form of doses of between 10⁴ and 10¹⁴ pfu/ml, andpreferably 10⁶ to 10¹⁰ pfu/ml. The term pfu (plaque forming unit)corresponds to the infectious power of a solution of virus, and isdetermined by infecting a suitable cell culture and measuring, generallyafter 5 days, the number of plaques of infected cells. The techniques ofdetermination of the pfu titre of a viral solution are well documentedin the literature.

The adenoviruses of the invention may be used for the treatment orprevention of a large number of pathologies. Depending on thetherapeutic gene inserted, the adenoviruses of the invention may beused, in particular, for the treatment or prevention of genetic diseases(dystrophy, cystic fibrosis, and the like), neurodegenerative diseases(Alzheimer's, Parkinson's, ALS, and the like), hyperproliferativepathologies (cancers, restenosis, and the like), pathologies associatedwith disorders of coagulation or with dyslipoproteinaemias, pathologiesassociated with viral infections (hepatitis, AIDS, and the like), andthe like.

The present invention will be described more completely by means of theexamples which follow, which should be considered to be illustrative andnonlimiting.

LEGENDS TO THE FIGURES

FIG. 1: Genetic organization of the Ad5 adenovirus. The completesequence of Ad5 is available on a database, and enables a person skilledin the art to select or create any restriction site, and thus to isolateany region of the genome.

FIG. 2: Restriction map of the CAV2 adenovirus strain Manhattan (fromSpibey et al., cited above).

FIG. 3: Construction of the vector pAD5-gp19k-βgal.

FIG. 4: Construction of the adenovirus Ad-gp19k-βgal, ΔE1,ΔE3.

GENERAL TECHNIQUES OF MOLECULAR BIOLOGY

The methods traditionally used in molecular biology, such as preparativeextractions of plasmid DNA, centrifugation of plasmid DNA in a caesiumchloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, phenol orphenol-chloroform extraction of proteins, ethanol or isopropanolprecipitation of DNA in a saline medium, transformation in Escherichiacoli, and the like, are well known to a person skilled in the art andare amply described in the literature [Maniatis T. et al., “MolecularCloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New York, 1987].

Plasmids of the pBR322 and pUC type and phages of the M13 series are ofcommercial origin (Bethesda Research Laboratories).

To carry out ligation, the DNA fragments may be separated according totheir size by agarose or acrylamide gel electrophoresis, extracted withphenol or with a phenol-chloroform mixture, precipitated with ethanoland then incubated in the presence of phage T4 DNA ligase (Biolabs)according to the supplier's recommendations.

The filling in of 5′ protruding ends may be performed with the Klenowfragment of E. coli DNA polymerase I (Biolabs) according to thesupplier's specifications. The destruction of 3′ protruding ends isperformed in the presence of phage T4 DNA polymerase (Biolabs) usedaccording to the manufacturer's recommendations. The destruction of 5′protruding ends is performed by a controlled treatment with S1 nuclease.

In vivo site-directed mutagenesis using synthetic oligodeoxynucleotidesmay be performed according to the method developed by Taylor et al.[Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed byAmersham.

The enzymatic amplification of DNA fragments by the so-called PCR[polymerase-catalysed chain reaction, Saiki R. K. et al., Science 230(1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155(1987) 335-350] technique may be performed using a “DNA thermal cycler”(Perkin Elmer Cetus) according to the manufacturer's specifications.

Verification of the nucleotide sequences may be performed by the methoddeveloped by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977)5463-5467] using the kit distributed by Amersham.

Cell Lines Used

In the examples which follow, the following cell lines have or may beused:

Human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977)59). This line contains, in particular, integrated in its genome, theleft-hand portion of the Ad5 human adenovirus genome(12%).

KB human cell line: Originating from a human epidermal carcinoma, thisline is available in the ATCC (ref. CCL17), together with the conditionsenabling it to be cultured.

Hela human cell line: Originating from a human epithelial carcinoma,this line is available in the ATCC (ref. CCL2), together with theconditions enabling it to be cultured.

MDCK canine cell line: The conditions of culture of MDCK cells have beendescribed, in particular, by Macatney et al., Science 44 (1988) 9.

gm DBP6 cell line (Brough et al., Virology 190 (1992) 624). This lineconsists of Hela cells carrying the adenovirus E2 gene under the controlof the MMTV LTR.

EXAMPLES Example 1.

Construction of defective recombinant adenoviruses comprising atherapeutic gene (the E. coli LacZ gene) under the control of the RSVLTRpromoter and the gp19k gene under the control of the RSVLTR promoter,both inserted in the E1 region.

These adenoviruses were constructed by homologous recombination betweena plasmid carrying the left-hand portion of the Ad5 adenovirus, the tworecombinant DNAs and a region of the Ad5 adenovirus (corresponding toprotein IX) and the DNA of a defective adenovirus carrying differentdeletions.

1. Construction of the Vector pAD5-gp19k-βgal (FIG. 3)

1.1. Construction of the Plasmid pGEM-gp19k

Plasmid pAD5-gp19k-βgal contains a cDNA sequence coding for theadenovirus gp19k protein. This plasmid was constructed as follows. TheXbaI fragment of the wild-type Ad5 adenovirus genome containing the E3region was isolated and cloned at the corresponding site of plasmid pGEM(Promega) to generate the plasmid pGEM-3. The HinfI fragment containingthe gp19k coding sequence (nucleotides 28628 to 29634 of the wild-typeAd5 adenovirus) was then isolated from plasmid pGEM-E3. The ends of thisfragment were rendered blunt by the action of the Klenow fragment ofE.coli DNA polymerase I (see General techniques of molecular biology),and the fragment obtained was then cloned at the Smal site of plasmidpGEMzf+ (Promega).

The plasmid obtained was designated pGEM-gp19k (FIG. 3).

1.2. Construction of the Vector pAD5-gp19k-βgal

This example describes the construction of a plasmid containing one ofthe two recombinant DNAs comprising their own promoter, the left-handportion of the adenovirus genome and an additional portion (protein pIX)permitting homologous recombination. This vector was constructed fromthe plasmid pAd.RSVβGal as follows.

Plasmid pAd.RSVβGal contains, in the 5′→3′ orientation,

the PvuII fragment corresponding to the left-hand end of the Ad5adenovirus, comprising: the ITR sequence, the origin of replication, theencapsidation signals and the E1A enhancer;

the gene coding for β-galactosidase under the control of the RSVpromoter (from Rous sarcoma virus),

a second fragment of the Ad5 adenovirus genome, which permits homologousrecombination between plasmid pAd.RSVβGal and the adenovirus d1324.Plasmid pAd.RSVβGal has been described by Stratford-Perricaudet et al.(J. Clin. Invest. 90 (1992) 626).

Plasmid pAd.RSVβGal was first cut with the enzymes EagI and ClaI. Thisgenerates a first fragment carrying, in particular, the left-handportion of the AdS adenovirus and the RSVLTR promoter. In parallel,plasmid pAD.RSVβGal was also cut with the enzymes EagI and XbaI. Thisgenerates a second type of fragment carrying, in particular, the RSVLTRpromoter, the LacZ gene and a fragment of the Ad5 adenovirus genomewhich permits homologous recombination. Clal-EagI and EagI-XbaIfragments were then ligated in the presence of the XbaI-ClaI fragment ofplasmid pGEM-gp19k (Example 1.1) carrying the gp19k coding sequence (seeFIG. 3). The vector thereby obtained, designated pAD5-gp19k-βgal, hencecontains

the PvuII fragment corresponding to the left-hand end of the AdSadenovirus comprising: the ITR sequence, the origin of replication, theencapsidation signals and the E1A enhancer;

the sequence coding for gp19k under the control of the RSV promoter(from Rous sarcoma virus);

the gene coding for β-galactosidase under the control of the RSVpromoter (from Rous sarcoma virus), and

a second fragment of the Ad5 adenovirus genome, which permits homologousrecombination.

2. Construction of Recombinant Adenoviruses

2.1. Construction of a recombinant adenovirus carrying a deletion in theE1 region, and carrying the two recombinant DNAs inserted in the sameorientation in the E1 region.

The vector pAD5-gp19k-βgal was linearized and cotransfected with anadenoviral vector deficient in the E1 gene into helper cells (line 293)supplying in trans the functions encoded by the adenovirus E1 (E1A andE1B) regions.

More specifically, the adenovirus Ad-gp19k-βgal,ΔE1 is obtained byhomologous recombination in vivo between the adenovirus Ad-RSVβgal (seeStratford-Perricaudet et al. cited above) and the vectorpAD5-gp19k-βgal, according to the following protocol: plasmidpAD5-gp19k-βgal, linearized with XmnI, and the adenovirus Ad-RSVβgal,linearized with the enzyme ClaI, are cotransfected into the line 293 inthe presence of calcium phosphate to permit homologous recombination.The recombinant adenoviruses thereby generated are then selected byplaque purification. After isolation, the DNA of the recombinantadenovirus is amplified in the cell line 293, leading to a culturesupernatant containing the unpurified defective recombinant adenovirushaving a titre of approximately 10¹⁰ pfu/ml.

The viral particles are generally purified by centrifugation on acaesium chloride gradient according to known techniques (see, inparticular, Graham et al., Virology 52 (1973) 456). The adenovirusAd-gp19k-βgal,ΔE1 may be stored at −80° C. in 20% glycerol.

2.2. Construction of a recombinant adenovirus carrying a deletion in theE1 and E3 regions, and carrying the two recombinant DNAs inserted in thesame orientation in the E1 region (FIG. 4).

The vector pAD5-gp19k-βgal was linearized and cotransfected with anadenoviral vector deficient in the E1 and E3 genes into helper cells(line 293) supplying in trans the functions encoded by the adenovirus E1(E1A and E1B) regions.

More specifically, the adenovirus Ad-gp19k-βgal,ΔE1,ΔE3 was obtained byhomologous recombination in vivo between the mutant adenovirus Ad-dl1324(Thimmappaya et al., Cell 31 (1982) 534) and the vector pAD5-gp19k-βgal,according to the following protocol: plasmid pAD5-gp19k-βgal and theadenovirus Ad-dl1324, the latter being linearized with the enzyme ClaI,were cotransfected into the line 293 in the presence of calciumphosphate to permit homologous recombination. The recombinantadenoviruses thereby generated were then selected by plaquepurification. After isolation, the DNA of the recombinant adenovirus wasamplified in the cell line 293, leading to a culture supernatantcontaining the unpurified defective recombinant adenovirus having atitre of approximately 10¹⁰ pfu/ml.

The viral particles are generally purified by centrifugation on acaesium chloride gradient according to known techniques (see, inparticular, Graham et al., Virology 52 (1973) 456). The genome of therecombinant adenovirus is then verified by Southern blot analysis. Theadenovirus Ad-gp19k-βgal,ΔE1,ΔE3 may be stored at −80° C. in 20% ofglycerol.

Example 2

Demonstration in Cell Culture of the Functionality of the Adenovirusesof the Invention

1. Transcription of gp19k in 3T3 Fibroblasts

The appearance of transcripts coding for gp19k in cells infected withthe adenovirus Ad-gp19k-βgal,ΔE1,ΔE3 was demonstrated by Northern blotanalysis of the total cellular RNAs. To this end, 5×10⁶ 3T3 cells wereinfected with 40 pfu/cell of virus. After 36 hours, total cellular RNAswere recovered by means of RNAzol (Cinna/Biotecx), precipitated and thenresuspended in water. 10 μg were then applied to formaldehyde gelcontaining 1.5% of agarose. The RNAs were then denatured in the presenceof 0.05 M NaOH, and thereafter transferred onto a nylon support(Hybond+, Amersham) by capillary transfer with 20×SSC. The nylonmembrane was prehybridized in medium comprising 6×SSC, 5×Denhardt's,0.5% SDS and 100 μg of denatured salmon sperm DNA for 2 h at 60° C. Aprobe corresponding to the DNA of the Ad5 adenovirus E3 region, labelledwith ³²P using the MegaPrime kit (Amersham), was then added to thesolution and left to hybridize overnight. After hybridization, themembrane was washed twice in a 2×SSC, 0.1% SDS medium at roomtemperature, then twice in a 0.1×SSC, 0.5% SDS medium at 45° C., thenlastly in a 0.1×SSC medium at room temperature, and exposed.

The results obtained show the appearance of a 1.6-kb band in cellsinfected with the adenovirus Ad-gp19k-βgal,ΔE1,ΔE3. This bandcorresponds to an mRNA comprising the sequence coding for gp19k andextending as far as the polyA site located in the RSV promotercontrolling the transcription of the LacZ gene. In contrast, noequivalent band is detected in cells infected with the adenovirusAd-βgal.

2. Expression of a Functional gp19k

The functionality of the gp19k produced by cells infected with theadenovirus Ad-gp19k-βgal,ΔE1,ΔE3 was verified by measuring, in an ELISAtest, the expression of β₂-microglobulin at the surface of the cells.β₂-Microglobulin is a nontransmembrane protein localized at the cellsurface in combination with MHC-I molecules, and which is needed forpresentation of the antigen at the cell surface. In particular, it isneeded for correct folding of the MHC-I molecules and for thepresentation of antigenic peptides at the cell surface, and constitutesas a result a good marker of the functionality of the MHC-I molecules.

3T3-Balb-c cells at confluence were infected with 200 pfu/cell ofAd-gp19k-βgal,ΔE1,ΔE3, and then incubated for 40 h at 37° C. under ahumid atmosphere, 5% CO₂, in DMEM medium containing 10% of foetal calfserum. The cells were then harvested in PBS buffer, 20 mM EDTA, andsuspended in DMEM medium, 10% FCS. 10⁵ cells were then introduced intoeach well of a 96-well plate. The plates were centrifuged at 250 g for 3min and then incubated for 6 hours. The wells were then washed with PBSbuffer, 1% bovine albumin (BSA) and thereafter incubated with a{fraction (1/500)} dilution of sheep anti-β₂-microglobulin antibody (TheBinding Site, Birmingham UK) in 1% BSA-PBS for 20 min at 37° C. Thecells were then washed in 1% BSA-PBS, thereafter fixed in PBS buffer,0.37% formaldehyde, 0.2% glutaraldehyde, washed twice and incubated witha {fraction (1/35,000)} dilution in 1% BSA-PBS of a 2nd anti-sheepantibody conjugated to alkaline phosphatase (AP) (Sigma) for 1 hour at4° C. The plates were then washed 3 times in 1% BSA-PBS. The AP activitywas then detected using the AP substrate kit (Biorad). The opticaldensity of the wells being read at 450 nm, the mean OD and the standarddeviation were calculated.

The results obtained show that infection with Ad-gp19k-βgal,ΔE1,ΔE3induces an approximately 40% reduction in the intensity of the surfaceβ₂-microglobulin signal revealed by the ELISA test, relative to theeffect produced by the adenovirus Ad-βgal. These results show clearlythat Ad-gp19k-βgal,ΔE1,ΔE3 induces the production of a functional gp19kprotein, and that the latter induces a significant fall in theexpression of MHC-I molecules at the cell surface.

Example 3

Immunoprotective Effect of the Adenoviruses of the Invention

3.1. Sensitivity to CTL Stimulated in Vivo with the Adenovirus Ad-βgal

This example shows that cells infected with the adenovirusAd-gp19k-βgal,ΔE1,ΔE3 are protected from lysis by CTL relative to cellsinfected with an Ad-βgal adenovirus.

DBA/2 mice (H-2d) received a first intravenous injection of 10⁸ pfu ofvirus Ad-βgal, followed 3 weeks later by a further intrapetitonealinjection of the same amount of virus. The mice were sacrificed 2 weekslater (at the earliest), their spleen was ground and the spleen cellswere suspended in 10 ml of RPMI 1640 (Gibco) containing 10% ofdecomplemented FCS, 50 μg/ml of streptomycin, 100 U/ml of penicillin,100 μg/ml of kanamycin and 100 μg/ml of gentamicin. The cells therebyobtained (CTL lymphocytes) were then incubated in the presence of 3T3cells infected with the adenovirus Ad-βgal. This leads to a lysis ofapproximately 40% of the stained (i.e. infected) cells. In contrast,these same cells (CTL lymphocytes) have practically no effect onuninfected fibroblasts, or on fibroblasts infected with the adenovirusAd-gp19k-βgal,ΔE1,ΔE3. These results show clearly that incorporation inthe vectors of the invention of a recombinant DNA capable of producinggp19k induces an immunoprotection of the infected cells.

3.2. Sensitivity to CTL Stimulated in Vivo with the AdenovirusAd-gp19k-βgal,ΔE1,ΔE3

This example shows that cells infected with the adenovirusAd-gp19k-βgal,ΔE1,ΔE3 are protected from lysis by CTL relative to cellsinfected with an Ad-βgal adenovirus.

Spleen cells of mice which had received injections ofAd-gp19k-βgal,ΔE1,ΔE3 were prepared under the conditions describedabove. Under the conditions described above, these CTL stimulated in thepresence of 3T3 cells infected with Ad-gp19k-βgal,ΔE1,ΔE3 do not inducelysis of the said cells, or of uninfected fibroblasts, or of fibroblastsinfected with Ad-βgal. Moreover, it was verified that this absence oflysis was not due to a poor viability of the cells. To this end, thesame lymphocytes were then stimulated in the presence of 3T3 cellsinfected with Ad-βgal, with the object of amplifying all the anti-βgalor antiadenovirus clones. The lymphocytes thereby obtained are capableof lysing 3T3 cells infected with Ad-βgal, in the same manner as thoseremoved from mice which had received Ad-βgal. These results show clearly(i) that the vectors of the invention induce an immunoprotection of theinfected cells, since no effective specific CTL is generated, and (ii)that while some anti-vector lymphocytes are generated in the spleens ofmice injected with Ad-gp19k-βgal,ΔE1,ΔE3, the expression of the antigensduring ex vivo stimulation is insufficient to induce a clonal expansionof these lymphocytes which is necessary for the lysis of the infectedtarget cells.

Example 4

Construction of defective recombinant adenoviruses comprising atherapeutic gene under the control of a promoter inserted in the E1region, and the gp19k gene under the control of the RSVLTR promoterinserted in the E3 region.

These adenoviruses were constructed by homologous recombination betweena the DNA of a first defective virus carrying the first recombinant DNA(therapeutic gene+promoter) inserted in the E1 region, and the DNA of asecond defective adenovirus carrying the second recombinant DNA(gp19k+RSV promoter) inserted in the E3 region.

1. Construction of the Defective Virus Carrying the Second RecombinantDNA (gp19k+RSV Promoter) Inserted in the E3 Region

This virus was constructed from the adenovirus Add1324 (Thimmappaya etal., Cell 31 (1982) 543). This virus carries a deletion in the E1 regionand in the E3 region (XbaI-EcoRI fragment deleted). The Add1324 virusDNA was isolated and purified. This DNA was then cut with the enzymesXbaI and EcoRI. An XbaI-EcoRI fragment was then obtained from plasmidpAd-gp19k-βgal carrying the sequence coding for gp19k under the controlof the RSV promoter, and thereafter inserted in the said sites into theAdd1324 DNA opened as before.

The DNA thereby obtained hence contains a deletion in the E1 region anda recombinant DNA in the E3 region carrying the gp19k gene under thecontrol of RSV.

2. Construction of Adenoviruses Carrying Both Recombinant DNAs

The DNA of the recombinant virus prepared above and the DNA of arecombinant adenovirus carrying a therapeutic gene in the E1 region, thelatter DNA being linearized with BamHI, are cotransfected into the line293 in the presence of calcium phosphate to permit homologousrecombination. The recombinant adenoviruses thereby generated are thenselected by plaque purification. After isolation, the DNA of therecombinant adenovirus is amplified in the cell line 293, leading to aculture supernatant containing the unpurified defective recombinantadenovirus having a titre of approximately 10¹⁰ pfu/ml.

The viral particles are generally purified by centrifugation on acaesium chloride gradient according to known techniques (see, inparticular, Graham et al., Virology 52 (1973) 456).

Although the examples above describe more especially the filled of thegp19k gene, it is to be understood that the approaches described in theexamples above may be repeated by a person skilled in the art usingother therapeutic genes, other immunoprotective genes, other promotersand other insertion sites in the adenovirus genome.

What is claimed is:
 1. A replication defective adenoviral vectorcomprising a first and second recombinant DNA sequence inserted into anadenoviral genome having a deleted or non-functional E3 region, whereinthe first recombinant DNA sequence can be expressed from the vector andwherein the second recombinant DNA sequence comprises the adenoviralimmunoprotective E3 gp19k coding region linked to a non-adenoviralpromoter, and wherein the adenoviral vector is capable of prolongedexpression of the first and second recombinant DNA sequences compared toa control adenovirus.
 2. The replication defective adenoviral vectoraccording to claim 1, wherein said first and second recombinant DNAsequences are inserted into the adenovirus genome in the sameorientation.
 3. The replication defective adenoviral vector according toclaim 1, wherein said first and second recombinant DNA sequences areinserted into the adenovirus genome in opposite orientations.
 4. Thereplication defective adenoviral vector according to claim 1, whereinsaid recombinant DNA sequences are inserted into the same region of theadenovirus genome.
 5. The replication defective adenoviral vectoraccording to claim 1, wherein the first and second recombinant DNAsequences are inserted into the adenovirus genome in the E1, E3 or E4region.
 6. The replication defective adenoviral vector according toclaim 1, wherein the first and second recombinant DNA sequences areinserted into the adenovirus genome in the E1 region.
 7. The replicationdefective adenoviral vector according to claim 1, wherein the first andsecond recombinant DNA sequences are inserted at different sites of theadenovirus genome and wherein the sites are selected from the groupconsisting of the adenovirus E1, E3, L5 and E4 regions.
 8. Thereplication defective adenoviral vector according to claim 1, whereinone recombinant DNA sequence is inserted into the adenovirus genome inthe E1 region and the other recombinant DNA sequence is inserted intothe adenovirus genome in the E3 or E4 region.
 9. The replicationdefective adenoviral vector according to claim 1, wherein the firstrecombinant DNA sequence is operably linked to a eukaryotic or viralpromoter.
 10. The replication defective adenoviral vector according toclaim 1, wherein the second recombinant DNA sequence comprises aconstitutive promoter.
 11. The replication defective adenoviral vectoraccording to claim 1, wherein the non-adenoviral promoter is a Roussarcoma virus LTR promoter.
 12. The replication defective adenoviralvector according to claim 1, wherein the second recombinant DNA is cDNA.13. The replication defective adenoviral vector according to claim 1,wherein the first and second recombinant DNA sequences constitute asingle transcriptional entity.
 14. The replication defective adenoviralvector according to claim 1, wherein the non-adenoviral promoter isselected from the group consisting of an MLP promoter, a CMV promoter,an RSV promoter, a PKG promoter, an albumin gene promoter, and acytokine-induced promoter.
 15. A replication defective adenoviral vectorcomprising a first and second recombinant DNA sequence inserted into anadenoviral genome having a deleted or non-functional E3 region, whereinthe first recombinant DNA sequence can be expressed from the vector andwherein the second recombinant DNA sequence comprises the herpes simplexvirus ICP47 coding region linked to a non-adenoviral promoter, andwherein the adenoviral vector is capable of prolonged expression of thefirst and second recombinant DNA sequences compared to a controladenovirus.
 16. The replication defective adenoviral vector according toclaim 15, wherein said first and second recombinant DNA sequences areinserted into the adenovirus genome in the same orientation.
 17. Thereplication defective adenoviral vector according to claim 15, whereinsaid first and second recombinant DNA sequences are inserted into theadenovirus genome in opposite orientations.
 18. The replicationdefective adenoviral vector according to claim 15, wherein saidrecombinant DNA sequences are inserted into the same region of theadenovirus genome.
 19. The replication defective adenoviral vectoraccording to claim 15, wherein the first and second recombinant DNAsequences are inserted into the adenovirus genome in the E1, E3 or E4region.
 20. The replication defective adenoviral vector according toclaim 15, wherein the first and second recombinant DNA sequences areinserted into the adenovirus genome in the E1 region.
 21. Thereplication defective adenoviral vector according to claim 15, whereinthe first and second recombinant DNA sequences are inserted at differentsites of the adenovirus genome and wherein the sites are selected fromthe group consisting of the adenovirus E1, E3, L5 and E4 regions. 22.The replication defective adenoviral vector according to claim 15,wherein one recombinant DNA sequence is inserted into the adenovirusgenome in the E1 region and the other recombinant DNA sequence isinserted into the adenovirus genome in the E3 or E4 region.
 23. Thereplication defective adenoviral vector according to claim 15, whereinthe first recombinant DNA sequence is operably linked to a eukaryotic orviral promoter.
 24. The replication defective adenoviral vectoraccording to claim 15, wherein the second recombinant DNA sequencecomprises a constitutive promoter.
 25. The replication defectiveadenoviral vector according to claim 15, wherein the non-adenoviralpromoter is a Rous sarcoma virus LTR promoter.
 26. The replicationdefective adenoviral vector according to claim 15, wherein the secondrecombinant DNA is cDNA.
 27. The replication defective adenoviral vectoraccording to claim 15, wherein the first and second recombinant DNAsequences constitute a single transcriptional entity.
 28. Thereplication defective adenoviral vector according to claim 15, whereinthe non-adenoviral promoter is selected from the group consisting of anMLP promoter, a CMV promoter, an RSV promoter, a PKG promoter, analbumin gene promoter, and a cytokine-induced promoter.